ABSTRACT Significance: Next generation sequencing (NGS) has has transformed the field of genomics and enabled broad adoption of the technology in biological, translational, and most recently clinical research. The proteomics equivalent to NGS has not yet been realized. As such, more accessible and cost-effective technologies for analysis of the proteome would accelerate biological and medical research. The goal of this proposal is to develop a highly-scalable digital protein analysis technology. The approach is broadly applicable and greatly improves upon cost, sensitivity, usability, and throughput of current approaches. Approach: We are developing a single molecule digital peptide sequencing platform using highly-parallelized single molecule Edman degradation and DNA encoding. Our assay generates a DNA library representing peptide sequence information on millions to billions of peptides that is sequenced on a standard NGS instrument. In this proposal, we are focused on developing reagents to expedite DNA tagging of the millions to billions of peptides for our assay by engineering proteases to cleave at modified sites. Impact: In summary, we believe our Next-Gen peptide sequencing assay will usher in the era of ?next gen proteomics? effectively transforming biological, translational, and clinical research, much like NGS technology has already done in the genomics space.